Nucleotide sequences which code for the tal gene

ABSTRACT

The invention relates to an isolated polynucleotide from coryneform bacteria, comprising a polynucleotide sequence chosen from the group consisting of  
     a) polynucleotide which is identical to the extent of at least 70% to a polynucleotide which codes for a polypeptide which comprises the amino acid sequences of SEQ ID NO. 2 or SEQ ID NO. 4,  
     b) polynucleotide which codes for a polypeptide which comprises an amino acid sequence which is identical to the extent of at least 70% to the amino acid sequences of SEQ ID NO. 2 or SEQ ID NO. 4  
     c) polynucleotide which is complementary to the polynucleotides of a) or b) and  
     d) polynucleotide comprising at least 15 successive nucleotides of the polynucleotide sequences of a), b) or c)  
     and a process for the preparation of L-amino acids, which comprises carrying out the following steps:  
     a) fermentation of the desired L-amino acid-producing bacteria in which at least the tal gene is amplified,  
     b) concentration of the desired product in the medium or in the cells of the bacteria and  
     c) isolation of the L-amino acid.

[0001] The invention provides nucleotide sequences which code for thetal gene and a process for the fermentative preparation of amino acids,in particular L-lysine, L-threonine, L-isoleucine and L-tryptophan,using coryneform bacteria in which the tal gene is amplified.

[0002] Prior Art

[0003] Amino acids, in particular L-lysine, are used in human medicineand in the pharmaceuticals industry, but in particular in animalnutrition.

[0004] It is known that amino acids are prepared by fermentation bystrains of coryneform bacteria, in particular Corynebacteriumglutamicum. Because of their great importance, work is constantly beingundertaken to improve the preparation processes. Improvements to theprocesses can relate to fermentation measures, such as e.g. stirring andsupply of oxygen, or the composition of the nutrient media, such as e.g.the sugar concentration during the fermentation, or the working up tothe product form by e.g. ion exchange chromatography, or the intrinsicoutput properties of the microorganism itself.

[0005] Methods of mutagenesis, selection and mutant selection are usedto improve the output properties of these microorganisms. Strains whichare resistant to antimetabolites, such as e.g. the lysine analogueS-(2-aminoethyl)-cysteine, or are auxotrophic for metabolites ofregulatory importance and produce L-amino acids, such as e.g. L-lysine,are obtained in this manner.

[0006] Methods of the recombinant DNA technique have also been employedfor some years for improving the strain of Corynebacterium strains whichproduce amino acids, by amplifying individual amino acid biosynthesisgenes and investigating the effect on the amino acid production. Reviewarticles in this context are to be found, inter alia, in Kinoshita(“Glutamic Acid Bacteria”, in: Biology of Industrial Microorganisms,Demain and Solomon (Eds.), Benjamin Cummings, London, UK, 1985,115-142), Hilliger (BioTec 2, 40-44 (1991)), Eggeling (Amino Acids6:261-272 (1994)), Jetten and Sinskey (Critical Reviews in Biotechnology15, 73-103 (1995)) and Sahm et al. (Annuals of the New York Academy ofScience 782, 25-39 (1996)).

[0007] The importance of the pentose phosphate cycle for thebiosynthesis and production of amino acids, in particular L-lysine, bycoryneform bacteria is the subject of numerous efforts among experts.

[0008] Thus Oishi and Aida (Agricultural and Biological Chemistry 29,83-89 (1965)) report on the “hexose monophosphate shunt” ofBrevibacterium ammoniagenes. Sugimoto and Shio (Agricultural andBilogical Chemistry 51, 101-108 (1987)) report on the regulation ofglucose 6-phosphate dehydrogenase in Brevibacterium flavum.

OBJECT OF THE INVENTION

[0009] The inventors had the object of providing new measures forimproved fermentative preparation of amino acids, in particularL-lysine, L-threonine, L-isoleucine and L-tryptophan.

DESCRIPTION OF THE INVENTION

[0010] Amino acids, in particular L-lysine, are used in human medicine,in the pharmaceuticals industry and in particular in animal nutrition.There is therefore a general interest in providing new improvedprocesses for the preparation of amino acids, in particular L-lysine.

[0011] When L-lysine or lysine are mentioned in the following, not onlythe base but also the salts, such as e.g. lysine monohydrochloride orlysine sulfate, are also meant by this.

[0012] The invention provides an isolated polynucleotide from coryneformbacteria, comprising a polynucleotide sequence chosen from the groupconsisting of

[0013] a) polynucleotide which is identical to the extent of at least70% to a polynucleotide which codes for a polypeptide which comprisesthe amino acid sequences of SEQ ID NO. 2 or SEQ ID NO. 4,

[0014] b) polynucleotide which codes for a polypeptide which comprisesan amino acid sequence which is identical to the extent of at least 70%to the amino acid sequences of SEQ ID NO. 2 or SEQ ID NO. 4,

[0015] c) polynucleotide which is complementary to the polynucleotidesof a) or b) and

[0016] d) polynucleotide comprising at least 15 successive nucleotidesof the polynucleotide sequence of a), b) or c).

[0017] The invention also provides the polynucleotide as claimed inclaim 1, this preferably being a DNA which is capable of replication,comprising:

[0018] (i) a nucleotide sequence chosen from the group consisting of SEQID NO. 1 and SEQ ID NO. 3 or

[0019] (ii) at least one sequence which corresponds to sequence (i)within the range of the degeneration of the genetic code, or

[0020] (iii) at least one sequence which hybridizes with the sequencecomplementary to sequence (i) or (ii), and optionally

[0021] (iv) sense mutations of neutral function in (i).

[0022] The invention also provides

[0023] a polynucleotide as claimed in claim 4, comprising one of thenucleotide sequences as shown in SEQ ID NO. 1 and SEQ ID NO. 3,

[0024] a polynucleotide as claimed in claim 5, which codes for apolypeptide which comprises the amino acid sequence as shown in SEQ IDNO. 2 and SEQ ID NO. 4,

[0025] a vector containing the polynucleotide as claimed in claim 1,

[0026] and coryneform bacteria, serving as the host cell, which containthe vector.

[0027] The invention also provides polynucleotides which substantiallycomprise a polynucleotide sequence, which are obtainable by screening bymeans of hybridization of a corresponding gene library, which comprisesthe complete gene with the polynucleotide sequence corresponding to SEQID NO. 1 or SEQ ID NO. 3, with a probe which comprises the sequence ofthe polynucleotide mentioned, according to SEQ ID NO. 1 or SEQ ID NO. 3or a fragment thereof, and isolation of the DNA sequence mentioned.

[0028] Polynucleotide sequences according to the invention are suitableas hybridization probes for RNA, cDNA and DNA, in order to isolate, inthe full length, cDNA which code for transaldolase and to isolate thosecDNA or genes which have a high similarity of sequence with that of thetransaldolase gene.

[0029] Polynucleotide sequences according to the invention arefurthermore suitable as primers for the preparation of DNA of geneswhich code for transaldolase by the polymerase chain reaction (PCR).

[0030] Such oligonucleotides which serve as probes or primers compriseat least 30, preferably at least 20, especially preferably at least 15successive nucleotides. Oligonucleotides which have a length of at least40 or 50 nucleotides are also suitable.

[0031] “Isolated” means separated out of its natural environment.

[0032] “Polynucleotide” in general relates to polyribonucleotides andpolydeoxyribonucleotides, it being possible for these to be non-modifiedRNA or DNA or modified RNA or DNA.

[0033] “Polypeptides” is understood as meaning peptides or proteinswhich comprise two or more amino acids bonded via peptide bonds.

[0034] The polypeptides according to the invention include a polypeptideaccording to SEQ ID NO. 2 or SEQ ID NO. 4, in particular those with thebiological activity of transaldolase, and also those which are identicalto the extent of at least 70% to the polypeptide according to SEQ ID NO.2 or SEQ ID NO. 4, and preferably are identical to the extent of atleast 80% and in particular to the extent of at least 90% to 95% to thepolypeptide according to SEQ ID NO. 2 or SEQ ID NO. 4, and have theactivity mentioned.

[0035] The invention also provides a process for the fermentativepreparation of amino acids, in particular L-lysine, L-threonine,L-isoleucine and L-tryptophan, using coryneform bacteria which inparticular already produce an amino acid, and in which the nucleotidesequences which code for the tal gene are amplified, in particularover-expressed.

[0036] The term “amplification” in this connection describes theincrease in the intracellular activity of one or more enzymes in amicroorganism which are coded by the corresponding DNA, for example byincreasing the number of copies of the gene or genes, using a potentpromoter or using a gene which codes for a corresponding enzyme having ahigh activity, and optionally combining these measures.

[0037] The microorganisms which the present invention provides canprepare L-amino acids, in particular L-lysine, from glucose, sucrose,lactose, fructose, maltose, molasses, starch, cellulose or from glyceroland ethanol. They can be representatives of coryneform bacteria, inparticular of the genus Corynebacterium. Of the genus Corynebacterium,there may be mentioned in particular the species Corynebacteriumglutamicum, which is known among experts for its ability to produceL-amino acids.

[0038] Suitable strains of the genus Corynebacterium, in particular ofthe species Corynebacterium glutamicum, are, for example, the knownwild-type strains

[0039]Corynebacterium glutamicum ATCC13032

[0040]Corynebacterium acetoglutamicum ATCC15806

[0041]Corynebacterium acetoacidophilum ATCC13870

[0042]Corynebacterium thermoaminogenes FERM BP-1539

[0043]Corynebacterium melassecola ATCC17965

[0044]Brevibacterium flavum ATCC14067

[0045]Brevibacterium lactofermentum ATCC13869 and

[0046]Brevibacterium divaricatum ATCC14020

[0047] and L-lysine-producing mutants or strains prepared therefrom,such as, for example

[0048]Corynebacterium glutamicum FERM-P 1709

[0049]Brevibacterium flavum FERM-P 1708

[0050]Brevibacterium lactofermentum FERM-P 1712

[0051]Corynebacterium glutamicum FERM-P 6463

[0052]Corynebacterium glutamicum FERM-P 6464 and

[0053]Corynebacterium glutamicum ATCC13032

[0054]Corynebacterium glutamicum DM58-1

[0055]Corynebacterium glutamicum DSM12866.

[0056] and L-threonine-producing mutants or strains prepared therefrom,such as, for example

[0057]Corynebacterium glutamicum ATCC21649

[0058]Brevibacterium flavum BB69

[0059]Brevibacterium flavum DSM5399

[0060]Brevibacterium lactofermentum FERM-BP 269

[0061]Brevibacterium lactofermentum TBB-10

[0062] and L-isoleucine-producing mutants or strains prepared therefrom,such as, for example

[0063]Corynebacterium glutamicum ATCC 14309

[0064]Corynebacterium glutamicum ATCC 14310

[0065]Corynebacterium glutamicum ATCC 14311

[0066]Corynebacterium glutamicum ATCC 15168

[0067]Corynebacterium ammoniagenes ATCC 6871

[0068] and L-tryptophan-producing mutants or strains prepared therefrom,such as, for example

[0069]Corynebacterium glutamicum ATCC21850 and

[0070]Corynebacterium glutamicum KY9218(pKW9901)

[0071] The inventors have succeeded in isolating the new tal gene of C.glutamicum which codes for transaldolase (EC 2.2.1.2).

[0072] To isolate the tal gene or also other genes of C. glutamicum, agene library of this microorganism is first set up in E. coli. Thesetting up of gene libraries is described in generally known textbooksand handbooks. The textbook by Winnacker: Gene und Klone, EineEinfuhrung in die Gentechnologie [Genes and Clones, An Introduction toGenetic Engineering] (Verlag Chemie, Weinheim, Germany, 1990) or thehandbook by Sambrook et al.: Molecular Cloning, A Laboratory Manual(Cold Spring Harbor Laboratory Press, 1989) may be mentioned as anexample. A well-known gene library is that of the E. coli K-12 strainW3110 set up in λ vectors by Kohara et al. (Cell 50, 495-508 (1987)).Bathe et al. (Molecular and General Genetics, 252:255-265, 1996)describe a gene library of C. glutamicum ATCC13032, which was set upwith the aid of the cosmid vector SuperCos I (Wahl et al., 1987,Proceedings of the National Academy of Sciences USA, 84:2160-2164) inthe E. coli K-12 strain NM554 (Raleigh et al., 1988, Nucleic AcidsResearch 16:1563-1575). Bormann et al. (Molecular Microbiology 6(3),317-326)) (1992)) in turn describe a gene library of C. glutamicumATCC13032 using the cosmid pHC79 (Hohn and Collins, Gene 11, 291-298(1980)). O'Donohue (The Cloning and Molecular Analysis of Four CommonAromatic Amino Acid Biosynthetic Genes from Corynebacterium glutamicum.Ph.D. Thesis, National University of Ireland, Galway, 1997) describesthe cloning of C. glutamicum genes using the λ Zap expression systemdescribed by Short et al. (Nucleic Acids Research, 16: 7583). To preparea gene library of C. glutamicum in E. coli it is also possible to useplasmids such as pBR322 (Bolivar, Life Sciences, 25, 807-818 (1979)) orpUC9 (Vieira et al., 1982, Gene, 19:259-268). Suitable hosts are, inparticular, those E. coli strains which are restriction- andrecombination-defective. An example of these is the strain DH5αmcr,which has been described by Grant et al. (Proceedings of the NationalAcademy of Sciences USA, 87 (1990) 4645-4649). The long DNA fragmentscloned with the aid of cosmids can then in turn be subcloned andsubsequently sequenced in the usual vectors which are suitable forsequencing, such as is described e.g. by Sanger et al. (Proceedings ofthe National Academy of Sciences of the United States of America,74:5463-5467, 1977).

[0073] The DNA sequences obtained can then be investigated with knownalgorithms or sequence analysis programs, such as e.g. that of Staden(Nucleic Acids Research 14, 217-232(1986)), the GCG program of Butler(Methods of Biochemical Analysis 39, 74-97 (1998)) the FASTA algorithmof Pearson and Lipman (Proceedings of the National Academy of SciencesUSA 85, 2444-2448 (1988)) or the BLAST algorithm of Altschul et al.(Nature Genetics 6, 119-129 (1994)) and compared with the sequenceentries which exist in databanks accessible to the public. Databanks fornucleotide sequences which are accessible to the public are, forexample, that of the European Molecular Biologies Laboratories (EMBL,Heidelberg, Germany) of that of the National Center for BiotechnologyInformation (NCBI, Bethesda, Md., USA).

[0074] The invention provides the new DNA sequence from C. glutamicumwhich contains the DNA section which codes for the tal gene, shown asSEQ ID NO 1 and SEQ ID NO 3. The amino acid sequence of thecorresponding protein has furthermore been derived from the present DNAsequence using the methods described above. The resulting amino acidsequence of the tal gene product is shown in SEQ ID NO 2 and SEQ ID NO4.

[0075] A gene library produced in the manner described above canfurthermore be investigated by hybridization with nucleotide probes ofknown sequence, such as, for example, the zwf gene (JP-A-09224661). Thecloned DNA of the clones which show a positive reaction in thehybridization is sequenced in turn to give on the one hand the knownnucleotide sequence of the probe employed and on the other hand theadjacent new DNA sequences.

[0076] Coding DNA sequences which result from SEQ ID NO 3 by thedegeneracy of the genetic code are also a constituent of the invention.In the same way, DNA sequences which hybridize with SEQ ID NO 3 or partsof or SEQ ID NO 3 are a constituent of the invention. Conservative aminoacid exchanges, such as e.g. exchange of glycine for alanine or ofaspartic acid for glutamic acid in proteins, are furthermore known amongexperts as “sense mutations” which do not lead to a fundamental changein the activity of the protein, i.e. are of neutral function. It isfurthermore known that changes on the N and/or C terminus of a proteincannot substantially impair or can even stabilize the function thereof.Information in this context can be found by the expert, inter alia, inBen-Bassat et al. (Journal of Bacteriology 169:751-757 (1987)), inO'Regan et al. (Gene 77:237-251 (1989)), in Sahin-Toth et al. (ProteinSciences 3:240-247 (1994)), in Hochuli et al. (Bio/Technology6:1321-1325 (1988)) and in known textbooks of genetics and molecularbiology. Amino acid sequences which result in a corresponding mannerfrom SEQ ID NO 2 or SEQ ID NO 4 are also a constituent of the invention.

[0077] In the same way, DNA sequences which hybridize with or SEQ ID NO3 or parts of or SEQ ID NO 3 are a constituent of the invention.Finally, DNA sequences which are prepared by the polymerase chainreaction (PCR) using primers which result from SEQ ID NO 3 are aconstituent of the invention. Such oligonucleotides typically have alength of at least 15 nucleotides.

[0078] Instructions for identifying DNA sequences by means ofhybridization can be found by the expert, inter alia, in the handbook“The DIG System Users Guide for Filter Hybridization” from BoehringerMannheim GmbH (Mannheim, Germany, 1993) and in Liebl et al.(International Journal of Systematic Bacteriology (1991) 41: 255-260).Instructions for amplification of DNA sequences with the aid of thepolymerase chain reaction (PCR) can be found by the expert, inter alia,in the handbook by Gait: Oligonukleotide synthesis: a practical approach(IRL Press, Oxford, UK, 1984) and in Newton and Graham: PCR (SpektrumAkademischer Verlag, Heidelberg, Germany, 1994).

[0079] The inventors have found that coryneform bacteria produce aminoacids in an improved manner after over-expression of the tal gene.

[0080] To achieve an over-expression, the number of copies of thecorresponding genes can be increased, or the promoter and regulationregion or the ribosome binding site upstream of the structural gene canbe mutated. Expression cassettes which are incorporated upstream of thestructural gene act in the same way. By inducible promoters, it isadditionally possible to increase the expression in the course offermentative L-amino acid production. The expression is likewiseimproved by measures to prolong the life of the m-RNA. Furthermore, theenzyme activity is also increased by preventing the degradation of theenzyme protein. The genes or gene constructs can either be present inplasmids with a varying number of copies, or can be integrated andamplified in the chromosome. Alternatively, an over-expression of thegenes in question can furthermore be achieved by changing thecomposition of the media and the culture procedure.

[0081] Instructions in this context can be found by the expert, interalia, in Martin et al. (Bio/Technology 5, 137-146 (1987)), in Guerreroet al. (Gene 138, 35-41 (1994)), Tsuchiya and Morinaga (Bio/Technology6, 428-430 (1988)), in Eikmanns et al. (Gene 102, 93-98 (1991)), inEuropean Patent Specification EPS 0 472 869, in U.S. Pat. No. 4,601,893,in Schwarzer and Puhler (Bio/Technology 9, 84-87 (1991), in Reinscheidet al. (Applied and Environmental Microbiology 60, 126-132 (1994)), inLaBarre et al. (Journal of Bacteriology 175, 1001-1007 (1993)), inPatent Application WO 96/15246, in Malumbres et al. (Gene 134, 15-24(1993)), in Japanese Laid-Open Specification JP-A-10-229891, in Jensenand Hammer (Biotechnology and Bioengineering 58, 191-195 (1998)), inMakrides (Microbiological Reviews 60:512-538 (1996)) and in knowntextbooks of genetics and molecular biology.

[0082] By way of example, the tal gene according to the invention wasover-expressed with the aid of plasmids.

[0083] Suitable plasmids are those which are replicated in coryneformbacteria. Numerous known plasmid vectors, such as e.g. pZ1 (Menkel etal., Applied and Environmental Microbiology (1989) 64: 549-554), pEKEx1(Eikmanns et al., Gene 102:93-98 (1991)) or pHS2-1 (Sonnen et al., Gene107:69-74 (1991)) are based on the cryptic plasmids pHM1519, pBL1 orpGA1. Other plasmid vectors, such as e.g. those based on pCG4 (U.S. Pat.No. 4,489,160), or pNG2 (Serwold-Davis et al., FEMS Microbiology Letters66, 119-124 (1990)), or pAG1 (U.S. Pat. No. 5,158,891), can be used inthe same manner.

[0084] Plasmid vectors which are furthermore suitable are also thosewith the aid of which the process of gene amplification by integrationinto the chromosome can be used, as has been described, for example, byReinscheid et al. (Applied and Environmental Microbiology 60, 126-132(1994)) for duplication or amplification of the hom-thrB operon. In thismethod, the complete gene is cloned in a plasmid vector which canreplicate in a host (typically E. coli), but not in C. glutamicum.Possible vectors are, for example, pSUP301 (Simon et al., Bio/Technology1, 784-791 (1983)), pK18mob or pK19mob (Schafer et al., Gene 145, 69-73(1994)), pGEM-T (Promega corporation, Madison, Wis., USA), pCR2.1-TOPO(Shuman (1994). Journal of Biological Chemistry 269:32678-84; U.S. Pat.No. 5,487,993), PCR® Blunt (Invitrogen, Groningen, Holland; Bernard etal., Journal of Molecular Biology, 234: 534-541 (1993)), pEM1 (Schrumpfet al, 1991, Journal of Bacteriology 173:4510-4516) or pBGS8 (Spratt etal., 1986, Gene 41: 337-342). The plasmid vector which contains the geneto be amplified is then transferred into the desired strain of C.glutamicum by conjugation or transformation. The method of conjugationis described, for example, by Schafer et al. (Applied and EnvironmentalMicrobiology 60, 756-759 (1994)). Methods for transformation aredescribed, for example, by Thierbach et al. (Applied Microbiology andBiotechnology 29, 356-362 (1988)), Dunican and Shivnan (Bio/Technology7, 1067-1070 (1989)) and Tauch et al. (FEMS Microbiological Letters 123,343-347 (1994)). After homologous recombination by means of a “crossover” event, the resulting strain contains at least two copies of thegene in question.

[0085] An example of a plasmid vector with the aid of which the processof amplification by integration can be carried out is pSUZ1, which isshown in FIG. 1. Plasmid pSUZ1 consists of the E. coli vector pBGS8described by Spratt et al. (Gene 41: 337-342(1986)), into which the talgene has been incorporated.

[0086] In addition, it may be advantageous for the production of aminoacids to amplify or over-express one or more enzymes of the particularbiosynthesis pathway, of glycolysis, of anaplerosis, of the pentosephosphate pathway or of amino acid export, in addition to the tal gene.

[0087] Thus, for example, for the preparation of L-amino acids, inparticular L-lysine, one or more genes chosen from the group consistingof

[0088] the dapA gene which codes for dihydrodipicolinate synthase (EP-B0 197 335),

[0089] the lysC gene which codes for a feed back resistant aspartatekinase (Kalinowski et al. (1990), Molecular and General Genetics 224:317-324),

[0090] the gap gene which codes for glycerolaldehyde 3-phosphatedehydrogenase (Eikmanns (1992), Journal of Bacteriology 174:6076-6086),

[0091] the pyc gene which codes for pyruvate carboxylase (DE-A-198 31609),

[0092] the mqo gene which codes for malate:quinone oxidoreductase(Molenaar et al., European Journal of Biochemistry 254, 395-403 (1998)),

[0093] the tkt gene which codes for transketolase (accession numberAB023377 of the databank of European Molecular Biologies Laboratories(EMBL, Heidelberg, Germany)),

[0094] the gnd gene which codes for 6-phosphogluconate dehydrogenase(JP-A-9-224662),

[0095] the zwf gene which codes for glucose 6-phosphate dehydrogenase(JP-A-9-224661),

[0096] the lysE gene which codes for lysine export (DE-A-195 48 222),

[0097] the zwal gene (DE 199 59 328.0; DSM 13115),

[0098] the eno gene which codes for enolase (DE: 19947791.4),

[0099] the devB gene,

[0100] the opcA gene (DSM 13264)

[0101] can be amplified, preferably over-expressed, at the same time.

[0102] Thus, for example, for the preparation of L-threonine, one ormore genes chosen from the group consisting of

[0103] at the same time the hom gene which codes for homoserinedehydrogenase (Peoples et al., Molecular Microbiology 2, 63-72 (1988))or the hom^(dr) allele which codes for a “feed back resistant”homoserine dehydrogenase (Archer et al., Gene 107, 53-59 (1991),

[0104] the gap gene which codes for glycerolaldehyde 3-phosphatedehydrogenase (Eikmanns (1992), Journal of Bacteriology 174:6076-6086),

[0105] the pyc gene which codes for pyruvate carboxylase (DE-A-198 31609),

[0106] the mqo gene which codes for malate:quinone oxidoreductase(Molenaar et al., European Journal of Biochemistry 254, 395-403 (1998)),

[0107] the tkt gene which codes for transketolase (accession numberAB023377 of the databank of European Molecular Biologies Laboratories(EMBL, Heidelberg, Germany)),

[0108] the gnd gene which codes for 6-phosphogluconate dehydrogenase(JP-A-9-224662),

[0109] the zwf gene which codes for glucose 6-phosphate dehydrogenase(JP-A-9-224661),

[0110] the thrE gene which codes for threonine export (DE 199 41 478.5;DSM 12840),

[0111] the zwal gene (DE 199 59 328.0; DSM 13115), the eno gene whichcodes for enolase (DE: 19947791.4),

[0112] the devB gene,

[0113] the opcA gene (DSM 13264)

[0114] can be amplified, preferably over-expressed, at the same time.

[0115] It may furthermore be advantageous for the production of aminoacids to attenuate

[0116] the pck gene which codes for phosphoenol pyruvate carboxykinase(DE 199 50 409.1 DSM 13047) and/or

[0117] the pgi gene which codes for glucose 6-phosphate isomerase (U.S.Ser. No. 09/396,478, DSM 12969), or

[0118] the poxB gene which codes for pyruvate oxidase (DE 199 51 975.7;DSM 13114), or

[0119] the zwa2 gene (DE: 199 59 327.2; DSM 13113) at the same time, inaddition to the amplification of the tal gene.

[0120] In addition to over-expression of the tal gene it may furthermorebe advantageous for the production of amino acids to eliminateundesirable side reactions (Nakayama: “Breeding of Amino Acid ProducingMicro-organisms”, in: Overproduction of Microbial Products, Krumphanzl,Sikyta, Vanek (eds.), Academic Press, London, UK, 1982).

[0121] The microorganisms prepared according to the invention can becultured continuously or discontinuously in the batch process (batchculture) or in the fed batch (feed process) or repeated fed batchprocess (repetitive feed process) for the purpose of production ofL-amino acids. A summary of known culture methods are described in thetextbook by Chmiel (Bioprozeβtechnik 1. Einfuhrung in dieBioverfahrenstechnik [Bioprocess Technology 1. Introduction toBioprocess Technology (Gustav Fischer Verlag, Stuttgart, 1991)) or inthe textbook by Storhas (Bioreaktoren und periphere Einrichtungen[Bioreactors and Peripheral Equipment] (Vieweg Verlag,Braunschweig/Wiesbaden, 1994)).

[0122] The culture medium to be used must meet the requirements of theparticular strains in a suitable manner. Descriptions of culture mediafor various microorganisms are contained in the handbook “Manual ofMethods for General Bacteriology” of the American Society forBacteriology (Washington D.C., USA, 1981). Sugars and carbohydrates,such as e.g. glucose, sucrose, lactose, fructose, maltose, molasses,starch and cellulose, oils and fats, such as e.g. soya oil, sunfloweroil, groundnut oil and coconut fat, fatty acids, such as e.g. palmiticacid, stearic acid and linoleic acid, alcohols, such as e.g. glyceroland ethanol, and organic acids, such as e.g. acetic acid, can be used asthe source of carbon. These substances can be used individually or as amixture. Organic nitrogen-containing compounds, such as peptones, yeastextract, meat extract, malt extract, corn steep liquor, soya bean flourand urea, or inorganic compounds, such as ammonium sulphate, ammoniumchloride, ammonium phosphate, ammonium carbonate and ammonium nitrate,can be used as the source of nitrogen. The sources of nitrogen can beused individually or as a mixture. Phosphoric acid, potassium dihydrogenphosphate or dipotassium hydrogen phosphate or the correspondingsodium-containing salts can be used as the source of phosphorus. Theculture medium must furthermore comprise salts of metals, such as e.g.magnesium sulfate or iron sulfate, which are necessary for growth.Finally, essential growth substances, such as amino acids and vitamins,can be employed in addition to the abovementioned substances. Suitableprecursors can moreover be added to the culture medium. The startingsubstances mentioned can be added to the culture in the form of a singlebatch, or can be fed in during the culture in a suitable manner.

[0123] Basic compounds, such as sodium hydroxide, potassium hydroxide,ammonia or aqueous ammonia, or acid compounds, such as phosphoric acidor sulfuric acid, can be employed in a suitable manner to control the pHof the culture. Antifoams, such as e.g. fatty acid polyglycol esters,can be employed to control the development of foam. Suitable substanceshaving a selective action, such as e.g. antibiotics, can be added to themedium to maintain the stability of plasmids. To maintain aerobicconditions, oxygen or oxygen-containing gas mixtures, such as e.g. air,are introduced into the culture. The temperature of the culture isusually 20° C. to 45° C., and preferably 25° C. to 40° C. Culturing iscontinued until a maximum of L-amino acid has formed. This target isusually reached within 10 hours to 160 hours.

[0124] The analysis of L-amino acids can be carried out by anionexchange chromatography with subsequent ninhydrin derivatization, asdescribed by Spackman et al. (Analytical Chemistry, 30, (1958), 1190).

[0125] The following microorganism has been deposited at the DeutscheSammlung für Mikrorganismen und Zellkulturen (DSMZ=German Collection ofMicroorganisms and Cell Cultures, Braunschweig, Germany) in accordancewith the Budapest Treaty:

[0126]Escherichia coli JM109/pSUZ1 as DSM 13263.

[0127] SEQ ID NO 1 also contains the new devB gene. The processaccording to the invention is used for fermentative preparation of aminoacids.

[0128] The following figures are attached:

[0129]FIG. 1: Map of the plasmid pSUZ1

[0130] The abbreviations and designations used have the followingmeaning. lacZ: segments of lacZα gene fragment kan r: kanamycinresistance tal: transaldolase gene ori: origin of replication of plasmidpBGS8 BclI: cleavage site of restriction enzyme BclI EcoRI: cleavagesite of restriction enzyme EcoRI HindIII: cleavage site of restrictionenzyme HindIII PstI: cleavage site of restriction enzyme PstI SacI:cleavage site of restriction enzyme SacI

EXAMPLES

[0131] The following examples will further illustrate this invention.The molecular biology techniques, e.g. plasmid DNA isolation,restriction enzyme treatment, ligations, standard transformations ofEscherichia coli etc. used are, (unless stated otherwise), described bySambrook et al., (Molecular Cloning. A Laboratory Manual (1989) ColdSpring Harbour Laboratories, USA).

Example 1

[0132] Preparation of a Genomic Cosmid Gene Library from Corynebacteriumglutamicum ATCC 13032

[0133] Chromosomal DNA from Corynebacterium glutamicum ATCC 13032 wasisolated as described by Tauch et al. (1995, Plasmid 33:168-179) andpartly cleaved with the restriction enzyme Sau3AI (Amersham Pharmacia,Freiburg, Germany, Product Description Sau3AI, Code no. 27-0913-02). TheDNA fragments were dephosphorylated with shrimp alkaline phosphatase(Roche Molecular Biochemicals, Mannheim, Germany, Product DescriptionSAP, Code no. 1758250). The DNA of the cosmid vector SuperCos1 (Wahl etal. (1987) Proceedings of the National Academy of Sciences USA84:2160-2164), obtained from Stratagene (La Jolla, USA, ProductDescription SuperCos1 Cosmid Vector Kit, Code no. 251301) was cleavedwith the restriction enzyme XbaI (Amersham Pharmacia, Freiburg, Germany,Product Description XbaI, Code no. 27-0948-O₂) and likewisedephosphorylated with shrimp alkaline phosphatase. The cosmid DNA wasthen cleaved with the restriction enzyme BamHI (Amersham Pharmacia,Freiburg, Germany, Product Description BamHI, Code no. 27-0868-04). Thecosmid DNA treated in this manner was mixed with the treated ATCC13032DNA and the batch was treated with T4 DNA ligase (Amersham Pharmacia,Freiburg, Germany, Product Description T4-DNA-Ligase, Codeno.27-0870-04). The ligation mixture was then packed in phages with theaid of Gigapack II XL Packing Extracts (Stratagene, La Jolla, USA,Product Description Gigapack II XL Packing Extract, Code no. 200217).For infection of the E. coli strain NM554 (Raleigh et al. 1988, NucleicAcid Research 16:1563-1575) the cells were taken up in 10 mM MgSO₄ andmixed with an aliquot of the phage suspension. The infection andtitering of the cosmid library were carried out as described by Sambrooket al. (1989, Molecular Cloning: A laboratory Manual, Cold SpringHarbor), the cells being plated out on LB agar (Lennox, 1955, Virology,1:190) with 100 μg/ml ampicillin. After incubation overnight at 37° C.,recombinant individual clones were selected.

Example 2

[0134] Isolation and Sequencing of the tal Gene

[0135] The cosmid DNA of an individual colony was isolated with theQiaprep Spin Miniprep Kit (Product No. 27106, Qiagen, Hilden, Germany)in accordance with the manufacturer's instructions and partly cleavedwith the restriction enzyme Sau3AI (Amersham Pharmacia, Freiburg,Germany, Product Description Sau3AI, Product No. 27-0913-02). The DNAfragments were dephosphorylated with shrimp alkaline phosphatase (RocheMolecular Biochemicals, Mannheim, Germany, Product Description SAP,Product No. 1758250). After separation by gel electrophoresis, thecosmid fragments in the size range of 1500 to 2000 bp were isolated withthe QiaExII Gel Extraction Kit (Product No. 20021, Qiagen, Hilden,Germany). The DNA of the sequencing vector pZero-1, obtained fromInvitrogen (Groningen, Holland, Product Description Zero BackgroundCloning Kit, Product No. K2500-01) was cleaved with the restrictionenzyme BamHI (Amersham Pharmacia, Freiburg, Germany, Product DescriptionBamHI, Product No. 27-0868-04). The ligation of the cosmid fragments inthe sequencing vector pZero-1 was carried out as described by Sambrooket al. (1989, Molecular Cloning: A laboratory Manual, Cold SpringHarbor), the DNA mixture being incubated overnight with T4 ligase(Pharmacia Biotech, Freiburg, Germany). This ligation mixture was thenelectroporated (Tauch et al. 1994, FEMS Microbiol Letters, 123:343-7)into the E. coli strain DH5αMCR (Grant, 1990, Proceedings of theNational Academy of Sciences U.S.A., 87:4645-4649) and plated out on LBagar (Lennox, 1955, Virology, 1:190) with 50 μg/ml zeocin. The plasmidpreparation of the recombinant clones was carried out with Biorobot 9600(Product No. 900200, Qiagen, Hilden, Germany). The sequencing wascarried out by the dideoxy chain-stopping method of Sanger et al. (1977,Proceedings of the National Academy of Sciences U.S.A., 74:5463-5467)with modifications according to Zimmermann et al. (1990, Nucleic AcidsResearch, 18:1067). The “RR dRhodamin Terminator Cycle Sequencing Kit”from PE Applied Biosystems(Product No. 403044, Weiterstadt, Germany) wasused. The separation by gel electrophoresis and analysis of thesequencing reaction were carried out in a “Rotiphoresis NFAcrylamide/Bisacrylamide” Gel (29:1) (Product No. A124.1, Roth,Karlsruhe, Germany) with the “ABI Prism 377” sequencer from PE AppliedBiosystems (Weiterstadt, Germany).

[0136] The raw sequence data obtained were then processed using theStaden program package (1986, Nucleic Acids Research, 14:217-231)version 97-0. The individual sequences of the pZero1 derivatives wereassembled to a continuous contig. The computer-assisted coding regionanalysis were prepared with the XNIP program (Staden, 1986, NucleicAcids Research, 14:217-231). Further analyses were carried out with the“BLAST search program” (Altschul et al., 1997, Nucleic Acids Research,25:3389-3402), against the non-redundant databank of the “NationalCenter for Biotechnology Information” (NCBI, Bethesda, Md., USA). Thenucleotide sequence obtained is shown in SEQ ID NO 1 and SEQ ID NO 3.

Example 3

[0137] Cloning of the tal Gene

[0138] PCR was used to amplify DNA fragments containing the entire talgene of C. glutamicum 13032 and flanking upstream and downstreamregions. PCR reactions were carried out using oligonucleotide primersdesigned from the sequence as determined in examples 1 and 2. GenomicDNA was isolated from Corynebacterium glutamicum ATCC13032 according toHeery and Dunican (Applied and Environmental Microbiology 59: 791-799(1993)) and used as template. The tal primers used were: fwd. primer:5′ GGT ACA AAG GGT CTT AAG 3′C rev. primer: 5′ GAT TTC ATG TCG CCG TTA3′

[0139] PCR Parameters were as follows:

[0140] 35 cycles

[0141] 95° C. for 3 minutes

[0142] 94° C. for 1 minute

[0143] 47° C. for 1 minute

[0144] 72° C. for 45 seconds

[0145] 2.0 mM MgCl2

[0146] approximately 150-200 ng DNA template.

[0147] The PCR product obtained was cloned into the commerciallyavailable pGEM-T vector purchased from Promega Corp. (pGEM-T Easy VectorSystem 1, cat. no. A1360, Promega UK, Southampton, UK) using strain E.coli JM109 (Yanisch-Perron et al., Gene, 33: 103-119 (1985)) as a host.The entire tal gene was subsequently isolated from the pGEM T-vector onan Eco RI fragment and cloned into the lacZQ EcoRI site of the E. colivector pBGS8 (Spratt et al., Gene 41(2-3): 337-342 (1986)). Therestriction enzymes used were obtained from Boehringer Mannheim UK Ltd.(Bell Lane, Lewes East Sussex BN7 1LG, UK) and used according tomanufacturer's instructions. E. coli JMI09 was then transformed withthis ligation mixture and electrotransformants were selected on Luriaagar supplemented with isopropyl-thiogalactopyranoside (IPTG),5-bromo-4-chloro-3-indolyl-galactopyranoside (XGAL) and kanamycin atconcentrations of 1 mM, 0.02% and 50 mg/l respectively. Plates wereincubated for twelve hours at 37° C. Plasmid DNA was isolated from onetransformant, characterised by restriction enzyme analysis using Eco RI.This new construct was designated pSUZ 1.

1 6 1 6995 DNA Corynebacterium glutamicum CDS (2471)..(3550) tal-Gen 1cacatttgaa ccacagttgg ttataaaatg ggttcaacat cactatggtt agaggtgttg 60acgggtcaga ttaagcaaag actactttcg gggtagatca cctttgccaa atttgaacca 120attaacctaa gtcgtagatc tgatcatcgg atctaacgaa aacgaaccaa aactttggtc 180ccggtttaac ccaggaagga ttgaccacct tgacgctgtc acctgaactt caggcgctca 240ctgtacgcaa ttacccctct gattggtccg atgtggacac caaggctgta gacactgttc 300gtgtcctcgc tgcagacgct gtagaaaact gtggctccgg ccacccaggc accgcaatga 360gcctggctcc ccttgcatac accttgtacc agcgggttat gaacgtagat ccacaggaca 420ccaactgggc aggccgtgac cgcttcgttc tttcttgtgg ccactcctct ttgacccagt 480acatccagct ttacttgggt ggattcggcc ttgagatgga tgacctgaag gctctgcgca 540cctgggattc cttgacccca ggacaccctg agtaccgcca caccaagggc gttgagatca 600ccactggccc tcttggccag ggtcttgcat ctgcagttgg tatggccatg gctgctcgtc 660gtgagcgtgg cctattcgac ccaaccgctg ctgagggcga atccccattc gaccaccaca 720tctacgtcat tgcttctgat ggtgacctgc aggaaggtgt cacctctgag gcatcctcca 780tcgctggcac ccagcagctg ggcaacctca tcgtgttctg ggatgacaac cgcatctcca 840tcgaagacaa cactgagatc gctttcaacg aggacgttgt tgctcgttac aaggcttacg 900gctggcagac cattgaggtt gaggctggcg aggacgttgc agcaatcgaa gctgcagtgg 960ctgaggctaa gaaggacacc aagcgaccta ccttcatccg cgttcgcacc atcatcggct 1020tcccagctcc aactatgatg aacaccggtg ctgtgcacgg tgctgctctt ggcgcagctg 1080aggttgcagc aaccaagact gagcttggat tcgatcctga ggctcacttc gcgatcgacg 1140atgaggttat cgctcacacc cgctccctcg cagagcgcgc tgcacagaag aaggctgcat 1200ggcaggtcaa gttcgatgag tgggcagctg ccaaccctga gaacaaggct ctgttcgatc 1260gcctgaactc ccgtgagctt ccagcgggct acgctgacga gctcccaaca tgggatgcag 1320atgagaaggg cgtcgcaact cgtaaggctt ccgaggctgc acttcaggca ctgggcaaga 1380cccttcctga gctgtggggc ggttccgctg acctcgcagg ttccaacaac accgtgatca 1440agggctcccc ttccttcggc cctgagtcca tctccaccga gacctggtct gctgagcctt 1500acggccgtaa cctgcacttc ggtatccgtg agcacgctat gggatccatc ctcaacggca 1560tttccctcca cggtggcacc cgcccatacg gcggaacctt cctcatcttc tccgactaca 1620tgcgtcctgc agttcgtctt gcagctctca tggagaccga cgcttactac gtctggaccc 1680acgactccat cggtctgggc gaagatggcc caacccacca gcctgttgaa accttggctg 1740cactgcgcgc catcccaggt ctgtccgtcc tgcgtcctgc agatgcgaac gagaccgccc 1800aggcttgggc tgcagcactt gagtacaagg aaggccctaa gggtcttgca ctgacccgcc 1860agaacgttcc tgttctggaa ggcaccaagg agaaggctgc tgaaggcgtt cgccgcggtg 1920gctacgtcct ggttgagggt tccaaggaaa ccccagatgt gatcctcatg ggctccggct 1980ccgaggttca gcttgcagtt aacgctgcga aggctctgga agctgagggc gttgcagctc 2040gcgttgtttc cgttccttgc atggattggt tccaggagca ggacgcagag tacatcgagt 2100ccgttctgcc tgcagctgtg accgctcgtg tgtctgttga agctggcatc gcaatgcctt 2160ggtaccgctt cttgggcacc cagggccgtg ctgtctccct tgagcacttc ggtgcttctg 2220cggattacca gaccctgttt gagaagttcg gcatcaccac cgatgcagtc gtggcagcgg 2280ccaaggactc cattaacggt taattgccct gctgttttta gcttcaaccc ggggcaatat 2340gattctccgg aattttattg ccccgggttg ttgttgttaa tcggtacaaa gggtcttaag 2400cacatccctt acttgcctgc tctccttgag cacagttcaa gaacaattct tttaaggaaa 2460atttagtttc atg tct cac att gat gat ctt gca cag ctc ggc act tcc 2509 MetSer His Ile Asp Asp Leu Ala Gln Leu Gly Thr Ser 1 5 10 act tgg ctc gacgac ctc tcc cgc gag cgc att act tcc ggc aat ctc 2557 Thr Trp Leu Asp AspLeu Ser Arg Glu Arg Ile Thr Ser Gly Asn Leu 15 20 25 agc cag gtt att gaggaa aag tct gta gtc ggt gtc acc acc aac cca 2605 Ser Gln Val Ile Glu GluLys Ser Val Val Gly Val Thr Thr Asn Pro 30 35 40 45 gct att ttc gca gcagca atg tcc aag ggc gat tcc tac gac gct cag 2653 Ala Ile Phe Ala Ala AlaMet Ser Lys Gly Asp Ser Tyr Asp Ala Gln 50 55 60 atc gca gag ctc aag gccgct ggc gca tct gtt gac cag gct gtt tac 2701 Ile Ala Glu Leu Lys Ala AlaGly Ala Ser Val Asp Gln Ala Val Tyr 65 70 75 gcc atg agc atc gac gac gttcgc aat gct tgt gat ctg ttc acc ggc 2749 Ala Met Ser Ile Asp Asp Val ArgAsn Ala Cys Asp Leu Phe Thr Gly 80 85 90 atc ttc gag tcc tcc aac ggc tacgac ggc cgc gtg tcc atc gag gtt 2797 Ile Phe Glu Ser Ser Asn Gly Tyr AspGly Arg Val Ser Ile Glu Val 95 100 105 gac cca cgt atc tct gct gac cgcgac gca acc ctg gct cag gcc aag 2845 Asp Pro Arg Ile Ser Ala Asp Arg AspAla Thr Leu Ala Gln Ala Lys 110 115 120 125 gag ctg tgg gca aag gtt gatcgt cca aac gtc atg atc aag atc cct 2893 Glu Leu Trp Ala Lys Val Asp ArgPro Asn Val Met Ile Lys Ile Pro 130 135 140 gca acc cca ggt tct ttg ccagca atc acc gac gct ttg gct gag ggc 2941 Ala Thr Pro Gly Ser Leu Pro AlaIle Thr Asp Ala Leu Ala Glu Gly 145 150 155 atc agc gtt aac gtc acc ttgatc ttc tcc gtt gct cgc tac cgc gag 2989 Ile Ser Val Asn Val Thr Leu IlePhe Ser Val Ala Arg Tyr Arg Glu 160 165 170 gtc atc gct gcg ttc atc gagggc atc aag cag gct gct gca aac ggc 3037 Val Ile Ala Ala Phe Ile Glu GlyIle Lys Gln Ala Ala Ala Asn Gly 175 180 185 cac gac gtc tcc aag atc cactct gtg gct tcc ttc ttc gtc tcc cgc 3085 His Asp Val Ser Lys Ile His SerVal Ala Ser Phe Phe Val Ser Arg 190 195 200 205 gtc gac gtt gag atc gacaag cgc ctc gag gca atc gga tcc gat gag 3133 Val Asp Val Glu Ile Asp LysArg Leu Glu Ala Ile Gly Ser Asp Glu 210 215 220 gct ttg gct ctg cgc ggcaag gca ggc gtt gcc aac gct cag cgc gct 3181 Ala Leu Ala Leu Arg Gly LysAla Gly Val Ala Asn Ala Gln Arg Ala 225 230 235 tac gct gtg tac aag gagctt ttc gac gcc gcc gag ctg cct gaa ggt 3229 Tyr Ala Val Tyr Lys Glu LeuPhe Asp Ala Ala Glu Leu Pro Glu Gly 240 245 250 gcc aac act cag cgc ccactg tgg gca tcc acc ggc gtg aag aac cct 3277 Ala Asn Thr Gln Arg Pro LeuTrp Ala Ser Thr Gly Val Lys Asn Pro 255 260 265 gcg tac gct gca act ctttac gtt tcc gag ctg gct ggt cca aac acc 3325 Ala Tyr Ala Ala Thr Leu TyrVal Ser Glu Leu Ala Gly Pro Asn Thr 270 275 280 285 gtc aac acc atg ccagaa ggc acc atc gac gcg gtt ctg gag cag ggc 3373 Val Asn Thr Met Pro GluGly Thr Ile Asp Ala Val Leu Glu Gln Gly 290 295 300 aac ctg cac ggt gacacc ctg tcc aac tcc gcg gca gaa gct gac gct 3421 Asn Leu His Gly Asp ThrLeu Ser Asn Ser Ala Ala Glu Ala Asp Ala 305 310 315 gtg ttc tcc cag cttgag gct ctg ggc gtt gac ttg gca gat gtc ttc 3469 Val Phe Ser Gln Leu GluAla Leu Gly Val Asp Leu Ala Asp Val Phe 320 325 330 cag gtc ctg gag accgag ggt gtg gac aag ttc gtt gct tct tgg agc 3517 Gln Val Leu Glu Thr GluGly Val Asp Lys Phe Val Ala Ser Trp Ser 335 340 345 gaa ctg ctt gag tccatg gaa gct cgc ctg aag tagaatcagc acgctgcatc 3570 Glu Leu Leu Glu SerMet Glu Ala Arg Leu Lys 350 355 360 agtaacggcg acatgaaatc gaattagttcgatcttatgt ggccgttaca catctttcat 3630 taaagaaagg atcgtgacac taccatcgtgagcacaaaca cgaccccctc cagctggaca 3690 aacccactgc gcgacccgca ggataaacgactcccccgca tcgctggccc ttccggcatg 3750 gtgatcttcg gtgtcactgg cgacttggctcgaaagaagc tgctccccgc catttatgat 3810 ctagcaaacc gcggattgct gcccccaggattctcgttgg taggttacgg ccgccgcgaa 3870 tggtccaaag aagactttga aaaatacgtacgcgatgccg caagtgctgg tgctcgtacg 3930 gaattccgtg aaaatgtttg ggagcgcctcgccgagggta tggaatttgt tcgcggcaac 3990 tttgatgatg atgcagcttt cgacaacctcgctgcaacac tcaagcgcat cgacaaaacc 4050 cgcggcaccg ccggcaactg ggcttactacctgtccattc caccagattc cttcacagcg 4110 gtctgccacc agctggagcg ttccggcatggctgaatcca ccgaagaagc atggcgccgc 4170 gtgatcatcg agaagccttt cggccacaacctcgaatccg cacacgagct caaccagctg 4230 gtcaacgcag tcttcccaga atcttctgtgttccgcatcg accactattt gggcaaggaa 4290 acagttcaaa acatcctggc tctgcgttttgctaaccagc tgtttgagcc actgtggaac 4350 tccaactacg ttgaccacgt ccagatcaccatggctgaag atattggctt gggtggacgt 4410 gctggttact acgacggcat cggcgcagcccgcgacgtca tccagaacca cctgatccag 4470 ctcttggctc tggttgccat ggaagaaccaatttctttcg tgccagcgca gctgcaggca 4530 gaaaagatca aggtgctctc tgcgacaaagccgtgctacc cattggataa aacctccgct 4590 cgtggtcagt acgctgccgg ttggcagggctctgagttag tcaagggact tcgcgaagaa 4650 gatggcttca accctgagtc caccactgagacttttgcgg cttgtacctt agagatcacg 4710 tctcgtcgct gggctggtgt gccgttctacctgcgcaccg gtaagcgtct tggtcgccgt 4770 gttactgaga ttgccgtggt gtttaaagacgcaccacacc agcctttcga cggcgacatg 4830 actgtatccc ttggccaaaa cgccatcgtgattcgcgtgc agcctgatga aggtgtgctc 4890 atccgcttcg gttccaaggt tccaggttctgccatggaag tccgtgacgt caacatggac 4950 ttctcctact cagaatcctt cactgaagaatcacctgaag catacgagcg cctcattttg 5010 gatgcgctgt tagatgaatc cagcctcttccctaccaacg aggaagtgga actgagctgg 5070 aagattctgg atccaattct tgaagcatgggatgccgatg gagaaccaga ggattaccca 5130 gcgggtacgt ggggtccaaa gagcgctgatgaaatgcttt cccgcaacgg tcacacctgg 5190 cgcaggccat aatttagggg caaaaaatgatctttgaact tccggatacc accacccagc 5250 aaatttccaa gaccctaact cgactgcgtgaatcgggcac ccaggtcacc accggccgag 5310 tgctcaccct catcgtggtc actgactccgaaagcgatgt cgctgcagtt accgagtcca 5370 ccaatgaagc ctcgcgcgag cacccatctcgcgtgatcat tttggtggtt ggcgataaaa 5430 ctgcagaaaa caaagttgac gcagaagtccgtatcggtgg cgacgctggt gcttccgaga 5490 tgatcatcat gcatctcaac ggacctgtcgctgacaagct ccagtatgtc gtcacaccac 5550 tgttgcttcc tgacaccccc atcgttgcttggtggccagg tgaatcacca aagaatcctt 5610 cccaggaccc aattggacgc atcgcacaacgacgcatcac tgatgctttg tacgaccgtg 5670 atgacgcact agaagatcgt gttgagaactatcacccagg tgataccgac atgacgtggg 5730 cgcgccttac ccagtggcgg ggacttgttgcctcctcatt ggatcaccca ccacacagcg 5790 aaatcacttc cgtgaggctg accggtgcaagcggcagtac ctcggtggat ttggctgcag 5850 gctggttggc gcggaggctg aaagtgcctgtgatccgcga ggtgacagat gctcccaccg 5910 tgccaaccga tgagtttggt actccactgctggctatcca gcgcctggag atcgttcgca 5970 ccaccggctc gatcatcatc accatctatgacgctcatac ccttcaggta gagatgccgg 6030 aatccggcaa tgccccatcg ctggtggctattggtcgtcg aagtgagtcc gactgcttgt 6090 ctgaggagct tcgccacatg gatccagatttgggctacca gcacgcacta tccggcttgt 6150 ccagcgtcaa gctggaaacc gtctaaggagaaatacaaca ctatggttga tgtagtacgc 6210 gcacgcgata ctgaagattt ggttgcacaggctgcctcca aattcattga ggttgttgaa 6270 gcagcaactg ccaataatgg caccgcacaggtagtgctca ccggtggtgg cgccggcatc 6330 aagttgctgg aaaagctcag cgttgatgcggctgaccttg cctgggatcg cattcatgtg 6390 ttcttcggcg atgagcgcaa tgtccctgtcagtgattctg agtccaatga gggccaggct 6450 cgtgaggcac tgttgtccaa ggtttctatccctgaagcca acattcacgg atatggtctc 6510 ggcgacgtag atcttgcaga ggcagcccgcgcttacgaag ctgtgttgga tgaattcgca 6570 ccaaacggct ttgatcttca cctgctcggcatgggtggcg aaggccatat caactccctg 6630 ttccctcaca ccgatgcagt caaggaatcctccgcaaagg tcatcgcggt gtttgattcc 6690 cctaagcctc cttcagagcg tgcaactctaacccttcctg cggttcactc cgcaaagcgc 6750 gtgtggttgc tggtttctgg tgcggagaaggctgaggcag ctgcggcgat cgtcaacggt 6810 gagcctgctg ttgagtggcc tgctgctggagctaccggat ctgaggaaac ggtattgttc 6870 ttggctgatg atgctgcagg aaatctctaagcagcgccag ctctaacaag aagctttaac 6930 aagaagctct aacgaaaagc actaacaaactaatccgggt gcgaaccttc atctgaatcg 6990 atgga 6995 2 360 PRTCorynebacterium glutamicum 2 Met Ser His Ile Asp Asp Leu Ala Gln Leu GlyThr Ser Thr Trp Leu 1 5 10 15 Asp Asp Leu Ser Arg Glu Arg Ile Thr SerGly Asn Leu Ser Gln Val 20 25 30 Ile Glu Glu Lys Ser Val Val Gly Val ThrThr Asn Pro Ala Ile Phe 35 40 45 Ala Ala Ala Met Ser Lys Gly Asp Ser TyrAsp Ala Gln Ile Ala Glu 50 55 60 Leu Lys Ala Ala Gly Ala Ser Val Asp GlnAla Val Tyr Ala Met Ser 65 70 75 80 Ile Asp Asp Val Arg Asn Ala Cys AspLeu Phe Thr Gly Ile Phe Glu 85 90 95 Ser Ser Asn Gly Tyr Asp Gly Arg ValSer Ile Glu Val Asp Pro Arg 100 105 110 Ile Ser Ala Asp Arg Asp Ala ThrLeu Ala Gln Ala Lys Glu Leu Trp 115 120 125 Ala Lys Val Asp Arg Pro AsnVal Met Ile Lys Ile Pro Ala Thr Pro 130 135 140 Gly Ser Leu Pro Ala IleThr Asp Ala Leu Ala Glu Gly Ile Ser Val 145 150 155 160 Asn Val Thr LeuIle Phe Ser Val Ala Arg Tyr Arg Glu Val Ile Ala 165 170 175 Ala Phe IleGlu Gly Ile Lys Gln Ala Ala Ala Asn Gly His Asp Val 180 185 190 Ser LysIle His Ser Val Ala Ser Phe Phe Val Ser Arg Val Asp Val 195 200 205 GluIle Asp Lys Arg Leu Glu Ala Ile Gly Ser Asp Glu Ala Leu Ala 210 215 220Leu Arg Gly Lys Ala Gly Val Ala Asn Ala Gln Arg Ala Tyr Ala Val 225 230235 240 Tyr Lys Glu Leu Phe Asp Ala Ala Glu Leu Pro Glu Gly Ala Asn Thr245 250 255 Gln Arg Pro Leu Trp Ala Ser Thr Gly Val Lys Asn Pro Ala TyrAla 260 265 270 Ala Thr Leu Tyr Val Ser Glu Leu Ala Gly Pro Asn Thr ValAsn Thr 275 280 285 Met Pro Glu Gly Thr Ile Asp Ala Val Leu Glu Gln GlyAsn Leu His 290 295 300 Gly Asp Thr Leu Ser Asn Ser Ala Ala Glu Ala AspAla Val Phe Ser 305 310 315 320 Gln Leu Glu Ala Leu Gly Val Asp Leu AlaAsp Val Phe Gln Val Leu 325 330 335 Glu Thr Glu Gly Val Asp Lys Phe ValAla Ser Trp Ser Glu Leu Leu 340 345 350 Glu Ser Met Glu Ala Arg Leu Lys355 360 3 1083 DNA Corynebacterium glutamicum CDS (1)..(1080) tal 3 atgtct cac att gat gat ctt gca cag ctc ggc act tcc act tgg ctc 48 Met SerHis Ile Asp Asp Leu Ala Gln Leu Gly Thr Ser Thr Trp Leu 1 5 10 15 gacgac ctc tcc cgc gag cgc att act tcc ggc aat ctc agc cag gtt 96 Asp AspLeu Ser Arg Glu Arg Ile Thr Ser Gly Asn Leu Ser Gln Val 20 25 30 att gaggaa aag tct gta gtc ggt gtc acc acc aac cca gct att ttc 144 Ile Glu GluLys Ser Val Val Gly Val Thr Thr Asn Pro Ala Ile Phe 35 40 45 gca gca gcaatg tcc aag ggc gat tcc tac gac gct cag atc gca gag 192 Ala Ala Ala MetSer Lys Gly Asp Ser Tyr Asp Ala Gln Ile Ala Glu 50 55 60 ctc aag gcc gctggc gca tct gtt gac cag gct gtt tac gcc atg agc 240 Leu Lys Ala Ala GlyAla Ser Val Asp Gln Ala Val Tyr Ala Met Ser 65 70 75 80 atc gac gac gttcgc aat gct tgt gat ctg ttc acc ggc atc ttc gag 288 Ile Asp Asp Val ArgAsn Ala Cys Asp Leu Phe Thr Gly Ile Phe Glu 85 90 95 tcc tcc aac ggc tacgac ggc cgc gtg tcc atc gag gtt gac cca cgt 336 Ser Ser Asn Gly Tyr AspGly Arg Val Ser Ile Glu Val Asp Pro Arg 100 105 110 atc tct gct gac cgcgac gca acc ctg gct cag gcc aag gag ctg tgg 384 Ile Ser Ala Asp Arg AspAla Thr Leu Ala Gln Ala Lys Glu Leu Trp 115 120 125 gca aag gtt gat cgtcca aac gtc atg atc aag atc cct gca acc cca 432 Ala Lys Val Asp Arg ProAsn Val Met Ile Lys Ile Pro Ala Thr Pro 130 135 140 ggt tct ttg cca gcaatc acc gac gct ttg gct gag ggc atc agc gtt 480 Gly Ser Leu Pro Ala IleThr Asp Ala Leu Ala Glu Gly Ile Ser Val 145 150 155 160 aac gtc acc ttgatc ttc tcc gtt gct cgc tac cgc gag gtc atc gct 528 Asn Val Thr Leu IlePhe Ser Val Ala Arg Tyr Arg Glu Val Ile Ala 165 170 175 gcg ttc atc gagggc atc aag cag gct gct gca aac ggc cac gac gtc 576 Ala Phe Ile Glu GlyIle Lys Gln Ala Ala Ala Asn Gly His Asp Val 180 185 190 tcc aag atc cactct gtg gct tcc ttc ttc gtc tcc cgc gtc gac gtt 624 Ser Lys Ile His SerVal Ala Ser Phe Phe Val Ser Arg Val Asp Val 195 200 205 gag atc gac aagcgc ctc gag gca atc gga tcc gat gag gct ttg gct 672 Glu Ile Asp Lys ArgLeu Glu Ala Ile Gly Ser Asp Glu Ala Leu Ala 210 215 220 ctg cgc ggc aaggca ggc gtt gcc aac gct cag cgc gct tac gct gtg 720 Leu Arg Gly Lys AlaGly Val Ala Asn Ala Gln Arg Ala Tyr Ala Val 225 230 235 240 tac aag gagctt ttc gac gcc gcc gag ctg cct gaa ggt gcc aac act 768 Tyr Lys Glu LeuPhe Asp Ala Ala Glu Leu Pro Glu Gly Ala Asn Thr 245 250 255 cag cgc ccactg tgg gca tcc acc ggc gtg aag aac cct gcg tac gct 816 Gln Arg Pro LeuTrp Ala Ser Thr Gly Val Lys Asn Pro Ala Tyr Ala 260 265 270 gca act ctttac gtt tcc gag ctg gct ggt cca aac acc gtc aac acc 864 Ala Thr Leu TyrVal Ser Glu Leu Ala Gly Pro Asn Thr Val Asn Thr 275 280 285 atg cca gaaggc acc atc gac gcg gtt ctg gag cag ggc aac ctg cac 912 Met Pro Glu GlyThr Ile Asp Ala Val Leu Glu Gln Gly Asn Leu His 290 295 300 ggt gac accctg tcc aac tcc gcg gca gaa gct gac gct gtg ttc tcc 960 Gly Asp Thr LeuSer Asn Ser Ala Ala Glu Ala Asp Ala Val Phe Ser 305 310 315 320 cag cttgag gct ctg ggc gtt gac ttg gca gat gtc ttc cag gtc ctg 1008 Gln Leu GluAla Leu Gly Val Asp Leu Ala Asp Val Phe Gln Val Leu 325 330 335 gag accgag ggt gtg gac aag ttc gtt gct tct tgg agc gaa ctg ctt 1056 Glu Thr GluGly Val Asp Lys Phe Val Ala Ser Trp Ser Glu Leu Leu 340 345 350 gag tccatg gaa gct cgc ctg aag tag 1083 Glu Ser Met Glu Ala Arg Leu Lys 355 3604 360 PRT Corynebacterium glutamicum 4 Met Ser His Ile Asp Asp Leu AlaGln Leu Gly Thr Ser Thr Trp Leu 1 5 10 15 Asp Asp Leu Ser Arg Glu ArgIle Thr Ser Gly Asn Leu Ser Gln Val 20 25 30 Ile Glu Glu Lys Ser Val ValGly Val Thr Thr Asn Pro Ala Ile Phe 35 40 45 Ala Ala Ala Met Ser Lys GlyAsp Ser Tyr Asp Ala Gln Ile Ala Glu 50 55 60 Leu Lys Ala Ala Gly Ala SerVal Asp Gln Ala Val Tyr Ala Met Ser 65 70 75 80 Ile Asp Asp Val Arg AsnAla Cys Asp Leu Phe Thr Gly Ile Phe Glu 85 90 95 Ser Ser Asn Gly Tyr AspGly Arg Val Ser Ile Glu Val Asp Pro Arg 100 105 110 Ile Ser Ala Asp ArgAsp Ala Thr Leu Ala Gln Ala Lys Glu Leu Trp 115 120 125 Ala Lys Val AspArg Pro Asn Val Met Ile Lys Ile Pro Ala Thr Pro 130 135 140 Gly Ser LeuPro Ala Ile Thr Asp Ala Leu Ala Glu Gly Ile Ser Val 145 150 155 160 AsnVal Thr Leu Ile Phe Ser Val Ala Arg Tyr Arg Glu Val Ile Ala 165 170 175Ala Phe Ile Glu Gly Ile Lys Gln Ala Ala Ala Asn Gly His Asp Val 180 185190 Ser Lys Ile His Ser Val Ala Ser Phe Phe Val Ser Arg Val Asp Val 195200 205 Glu Ile Asp Lys Arg Leu Glu Ala Ile Gly Ser Asp Glu Ala Leu Ala210 215 220 Leu Arg Gly Lys Ala Gly Val Ala Asn Ala Gln Arg Ala Tyr AlaVal 225 230 235 240 Tyr Lys Glu Leu Phe Asp Ala Ala Glu Leu Pro Glu GlyAla Asn Thr 245 250 255 Gln Arg Pro Leu Trp Ala Ser Thr Gly Val Lys AsnPro Ala Tyr Ala 260 265 270 Ala Thr Leu Tyr Val Ser Glu Leu Ala Gly ProAsn Thr Val Asn Thr 275 280 285 Met Pro Glu Gly Thr Ile Asp Ala Val LeuGlu Gln Gly Asn Leu His 290 295 300 Gly Asp Thr Leu Ser Asn Ser Ala AlaGlu Ala Asp Ala Val Phe Ser 305 310 315 320 Gln Leu Glu Ala Leu Gly ValAsp Leu Ala Asp Val Phe Gln Val Leu 325 330 335 Glu Thr Glu Gly Val AspLys Phe Val Ala Ser Trp Ser Glu Leu Leu 340 345 350 Glu Ser Met Glu AlaArg Leu Lys 355 360 5 18 DNA Artificial Sequence Description ofArtificial Sequence Primer 5 ggtacaaagg gtcttaag 18 6 18 DNA ArtificialSequence Description of Artificial Sequence Primer 6 gatttcatgt cgccgtta18

1-7. Canceled
 8. A process for the preparation of L-amino acids, whichcomprises carrying out the following steps: a) fermentation of thebacteria which produce the desired L-amino acid, in which at least thetal gene and optionally one or more of the genes tkt gene, zwt gene,devB gene or opcA gene are amplified at the same time, b) concentrationof the desired product in the medium or in the cells of the bacteria andc) isolation of the desired L-amino acid.
 9. A process as claimed inclaim 8, wherein bacteria in which further genes of the biosynthesispathway of the desired L-amino acid are additionally amplified areemployed.
 10. A process as claimed in claim 9, wherein bacteria in whichthe metabolic pathways which reduce the formation of the desired L-aminoacid are at least partly eliminated are employed.
 11. A process asclaimed in claim 10, wherein coryneform bacteria which produce one ofthe amino acids from the group consisting of L-lysine, L-threonine,L-isoleucine or L-tryptophan are used.
 12. A process for thefermentative preparation of L-amino acids, in particular lysine, asclaimed in claim 8, wherein in the coryneform microorganisms which inparticular already produce L-amino acids, one or more genes chosen fromthe group consisting of 12.1 the dapA gene which codes fordihydrodipicolinate synthase, 12.2 the lysC gene which codes for a feedback resistant aspartate kinase, 12.3 the gap gene which codes forglycerolaldehyde 3-phosphate dehydrogenase, 12.4 the pyc gene whichcodes for pyruvate carboxylase, 12.5 the mqo gene which codes formalate-quinone oxidoreductase, 12.6 the tkt gene which codes fortransketolase, 12.7 the gnd gene which codes for 6-phosphogluconatedehydrogenase, 12.8 the zwf gene which codes for glucose 6-phosphatedehydrogenase, 12.9 the lysE gene which codes for lysine export, 12.10the zwal gene, 12.11 the eno gene which codes for enolase, 12.12 theopcA gene is or are amplified or over-expressed at the same time.
 13. Aprocess for the fermentative preparation of L-threonine as claimed inclaim 8, wherein in coryneform microorganisms which in particularalready produce L-threonine, one or more genes chosen from the groupconsisting of 13.1 at the same time the hom gene which codes forhomoserine dehydrogenase or the hom^(dr) allele which codes for a “feedback resistant” homoserine dehydrogenase, 13.2 the gap gene which codesfor glyceraldehyde 3-phosphate drhydrogenase, 13.3 the pyc gene whichcodes for pyruvate carboxylase, 13.4 the mqo gene which codes formalate:quinone oxidoreductase, 13.5 the tkt gene which codes fortransketolase, 13.6 the gnd gene which codes for 6-phosphogluconatedehydrogenase, 13.7 the zwf gene which codes for glucose 6-phosphatedehydrogenase, 13.8 the thrE gene which codes for threonine export, 13.9the zwal gene, 13.10 the eno gene which codes for enolase, 13.11 theopcA gene is or are amplified, in particular over-expressed, at the sametime.
 14. A process as claimed in claim 10, wherein for the preparationof L-amino acids, in particular L-lysine, L-threonine, L-isoleucine orL-tryptophan, bacteria in which one or more genes chosen from the groupconsisting of, 14.1 the pck gene which codes for pyhosphoenol pyruvatecarbosykinase, 14.2 the pgi gene which codes for glucose 6-phosphate6isomerase 14.3 the poxB gene which codes for pyruvate oxidase or 14.4the zwa2 gene is or are attenuated at the same time, are fermented. 15.Canceled
 16. Canceled